Establishment Of LAMP Detection Method And Study Onmolecular Vaccines Co-expressing Two Genes Of Brucellosis

Brucellosis, caused by Brucella spp., is a highly infectious zoonotic disease occurring in humans andvarious species of domesticated and wild animals worldwide. At present, the incidence of human andanimal brucellosis trends to be increasing. It causes a great economic impact on livestock industry andthreats to human health. It is urgent to prevent and control the disease with new diagnostic methods andnew generation vaccines with high efficacy and safety. The main contents of this study are as thefollowing:1. Complete coding sequences of Brucella genes OMP25, P39,18KD, L7/L12and BCSP31wereamplified and cloned into pMD18-T vector, and a series of cloning plasmids were verified by sequencesanalysis. Then based on reported OMP25sequence, two sets of four LAMP primers were designed withonline softwares. Through the optimization of the reaction system and conditions, a novel LAMP assaywas established to detect Brucella DNA from six classical species. It was confirmed that the assay wasable to detect a limit9fg/ml Brucella DNA with good sensitivity, which was10times higher than thatof a reference nested PCR. The results in detection of field samples showed that the LAMP assaydeveloped in this study yielded a99.0%concordance rate in comparison with nested PCR. These resultsindicate that the LAMP assay is a potential for the diagnosis of Brucellosis with high convenience,rapidity, sensitivity and specificity.2. Using pMD18-T, pQCXIX and pcDNA3.1, we successfully constructed a recombinant DNAvaccine pcDNA-P39/18KD with IRES sequence. The pcDNA-P39/18KD recombinant plasmids weretransfected into293AD cells using lipofectamine. The insertion of two genes was validated by RT-PCR,IFA and Western blot. The co-expression of P39and18KD genes cloned into pcDNA3.1was confirmedand its specific reactivity with antisera was observed.3. L7/L12and BCSP31genes were excised from pQCXIX vector with IRES sequence and clonedinto a transfer vector pShuttle-CMV. The recombinant plasmid pSh-LL/BP was constructed and verifiedby sequence analysis. Through homologous recombination between the plasmid pSh-LL/BP andskeleton vector pAdEasy-1in E. coli BJ5183, a recombinant plasmid pAd-LL/BP was obtained. Then293AD cells were successfully transfected and a recombinant adenovirus Ad-LL/BP was packed. Thetiter of the recombinant adenovirus containing L7/L12and BCSP31genes of Brucella spp. was109.68TCID50/mL in293AD monolayer cells. After continuously passaging, maintenance and stableco-expression of the two target genes in the recombinant adenovirus Ad-LL/BP were confirmed by PCR,IFA, Western blot and transmission electron microscope, and the two target proteins showed specificreactivity.4. The pcDNA-P39/18KD DNA vaccine and the pAd-LL/BP adenoviral vector vaccine wereevaluated in BALB/c mice by intramuscular injection alone or in combination after proliferated andpurified. Fifty BALB/c mice were divided into ten groups (5mice in each group), and inoculated threetimes at a two-week interval. Blood samples were taken before each injection, and15days after final administration the mice were sacrificed for blood and aplenocytes collection.ELISA results showed that the IgG was elicited in serum of all the mice immunized with DNA andrecombinant adenovirus vaccines alone or in combination from the15th-day post first inoculation. Inprime-boost, combination immunization groups showed significantly higher antibody levels than eithervaccine alone. The highest level of specific IgG was observed in the group with pcDNA-P39/18KD(prime) and Ad-LL/BP (boost) regimen, and the lowest in the group with pcDNA-P39/18KDimmunization. Except for IgA, the levels of all IgG, IgG1and IgG2a all rose with the increase ofvaccination times, in which IgG2a levels were great higher than IgG1(p<0.05). MTT results showedthat lympho-spleencytes stimulation index (SI) of the mice in each group immunized with DNA vaccineand recombinant adenovirus vaccine alone or in combination increased after A19antigen or ConAstimulation, but a little higher with ConA stimulation than A19antigen (p>0.05). A SI order in differentgroups was Cp>V3>V4>V1>V2>Cn. Analysis of spleen T lymphocyte subsets by FCM showed that thepercentage of CD³⁺and CD⁴⁺from mice was significantly higher in immunized groups than in negativecontrol groups, and also higher in prime-boost combination immunization groups than each vaccinealone. But there were no remarkable differences in the percentage of CD⁸⁺and CD⁴⁺/CD⁸⁺from mice indifferent immunized groups. ELISA results of the induced IL-12levels from spleen lymphocytesshowed that the expression levels of IL-12were70.7±8.1pg/mL and64.2±7.5pg/mL in DNA/Ad andAd/DNA combination immunization group, respectively, which were significantly higher than36.4±4.4pg/mL and38.7±5.15pg/mL in respective DNA and recombinant adenovirus immunization alone(p<0.05). Conversely, there were no obvious differences in IL-10levels between the immunized groupsand the negative control groups.In summary, the DNA and recombinant adenovirus vaccines co-expressing two genes of Brucella spp.constructed in this study were able not only to induce high level IgG specific antibody, but also toinduce both specific humoral immunity and predominant Th1-type cellular immune responses inBALB/c mice. The present study is the first demonstration that Brucella-specific immune responses canbe enhanced more significantly using prime-boost combination immunization than either the DNAvaccine or the recombinant adenovirus vaccine. The immune response induced by thepcDNA-P39/18KD priming and the Ad-LL/BP boosting appeared to be efficient against Brucellosis inmouse.